1. Field of the Invention
This invention relates to new methods of synthesizing mononucleoside H-phosphonates and oligonucleotides using the H-phosphonate method.
2. Summary of the Related Art
There has been much interest in recent years in the use of antisense oligonucleotides as instruments for the selective modulation of gene expression in vitro and in vivo. E.g., Agrawal, Trends in Biotech. 10, 152 (1992); Chang and Petit, Prog. Biophys. Molec. Biol. 58, 225 (1992). Antisense oligonucleotides are constructed to be sufficiently complementary to a target nucleic acid to hybridize with the target under the conditions of interest and inhibit expression of the target. Antisense oligonucleotides may be designed to bind directly to DNA (the so-called "anti-gene" approach) or to mRNA. Id. Expression inhibition is believed to occur by prevention of transcription or translation, or inducement of target mRNA cleavage by RNase H.
Antisense oligonucleotides can be used as a research tool in vitro to determine the biological function of genes and proteins. They provide an easily used alternative to the laborious method of gene mutation (e.g., deletion mutation) to selectively inhibit gene expression. The importance of this method is readily appreciated when one realizes that the elucidation of all known biological processes was determined by deletion mutation.
Antisense oligonucleotides also may be used to treat a variety of pathogenic diseases by inhibiting nucleic acid expression of the pathogen in vivo.
Simple methods for synthesizing and purifying oligonucleotides are now in great demand due to the utility of synthetic oligonucleotides in a wide variety of molecular biological techniques. Initially, the method of choice for synthesizing oligonucleotides was the beta-cyanoethyl phosphoramidite method. Beaucage and Caruthers, Tetrahedron Lett. 22, 1859 (1981). In the phosphoramidite procedure, the first nucleotide (monomer 1) is bound by its 3'-hydroxyl to a solid matrix while its 5'-hydroxyl remains available for binding. The synthesis of the first internucleotide link is carried out by mixing bound monomer 1 with a second nucleotide that has a reactive 3'-diisopropyl phosphoramidite group on its 3'-hydroxyl and a blocking group on its 5'-hydroxyl (monomer 2). In the presence of a weak acid, coupling of monomer 1 and monomer 2 occurs as a phosphodiester with the phosphorus in a trivalent state. This is oxidized, giving a pentavalent phosphotriester. The protecting group is then removed from monomer 2 and the process is repeated.
The H-phosphonate approach was first reported by Hale et al., J. Chem. Soc., 3291 (1957) and revisited some twenty years later by Sekine and Hata, Tetrahedron Lett. 16, 1711 (1975), Sekine et al., Tetrahedron Lett. 20, 1145 (1979), Garegg et al., Chemica Scripta 25, 280 (1985) ("Garegg I"), and Garegg et al., Chemica Scripta 26, 59 (1986) ("Garegg II"). The H-phosphonate method involves condensing the 5' hydroxyl group of the nascent oligonucleotide with a nucleoside having a 3' phosphonate moiety. Once the entire chain is constructed, the phosphite diester linkages are oxidized with t-butyl hydroperoxide or iodine to yield the corresponding phosphotriester. See, e.g., Froehler, "Oligodeoxynucleotide Synthesis," in Methods in Molecular Biology, Vol. 20, Protocols for Oligonucleotides and Analogs, p. 63-80 (S. Agrawal, Ed., Humana Press 1993) ("Froehler I"); Uhlmann and Peyman, Chem. Rev. 90, 543 (1990).
The H-phosphonate approach became practical only with the introduction of pivaloyl chloride as the condensing agent. Sterically hindered carbonyl chlorides such as adamantoyl and pivaloyl chloride (trimethyl acetyl chloride) are typically used as condensing agents. U.S. Pat. No. 4,959,463; European Pat. App. 86307926.5. Since then there have been reports of successful use of this method in both deoxyribonucleotide (Garegg et al., Tetrahedron Lett., 27, 4051 (1986) ("Garegg III"); Froehler et al., Nucl. Acid. Res. 14, 5399 (1986) ("Froehler II")) and ribonucleotide syntheses (Garegg et al., Tetrahedron Lett. 27, 4055 (1986) ("Garegg IV")). See generally Stawinski, "Some Aspects of H-Phosphonate Chemistry," in Handbook of Organophosphorus Chemistry, pp. 377-434 (R. Engel, Ed., Marcel Dekker, Inc., New York 1992). The H-phosphonate method offers several advantages over the beta-cyanoethyl phosphoramidite method. The 3'-phosphonate monomers are easily prepared and are stable to hydrolysis and oxidation. H-phosphonate chemistry requires no phosphate protecting group because phosphite diester linkages are relatively inert to coupling conditions. Furthermore, the H-phosphonate method requires a shorter cycle time. Finally, a simple reaction can be used to prepare backbone-modified DNA and RNA from the H-phosphonate synthesis product.
The H-phosphonate methods of Froehler I, Garegg III, and Garegg IV, supra, although adequate for small scale synthesis (i.e., less than 1 .mu.mol), are not practical on a large scale (e.g., 10-20 .mu.mol). The main reason is that the methods reported by these groups require 20-30 equivalents of monomer per coupling reaction. At this rate, the monomer consumption costs represent approximately 60% of the oligonucleotide assembly cost.
Gaffney et al., Tetrahedron Lett. 29, 2619 (1988), reported an effort to scale up H-phosphonate oligonucleotide synthesis to the 10-20 .mu.mol range while reducing the monomer equivalents consumed per coupling reaction. In synthesizing an 8-mer (consuming 1.53 equivalents of H-phosphonate) and a 26-mer (consuming 5.5 equivalents of H-phosphonate), however, Gaffney's group reported an average coupling yield of only 81% and 87%, respectively. Because of this relatively low coupling efficiency as compared with prior art methods, the authors found it necessary to employ a separate capping step using cyanoethyl H-phosphonate to prevent the elongation of truncated failed sequences in subsequent synthetic cycles. This extra step was necessary because the self-capping efficiency for pivaloyl chloride was found to be too low. According to the method of Gaffney et al., which assumed a 94% coupling yield, the expected result of a 20-mer synthetic reaction would be a crude mixture consisting of 24% product (20-mer) and 76% short chains (e.g., 19-mers, 18-mers, etc.).
Decreased yields are due in large part to some side reactions between the condensing agent and starting material. Efimov et al., Nucl. Acids Res. 21, 5337 (1993), demonstrated the use of dipentafluorophenyl carbonate as an activating agent for the H-phosphonate reaction. Use of this compound resulted in a high coupling efficiency with a concomitant decrease in side reactions.
A variety of methods of preparing mononucleoside H-phosphonates have been proposed, including PCl.sub.3 /azole system (Garegg II, supra; Froehler II, supra), salicylchlorophosphite (Marugg et al., Tetrahedron Lett. 27, 2661 (1986)), di- and tri(2,2,2-trifluoroethyl) H-phosphonates (Gibbs and Larsen, Synthesis-Stuttgart, pp. 410-413 (1984), pyro-H-phosphonate (Sakatsume et al., Nucleic Acids Res. 17, 3689 (1989), and transesterification of diphenyl H-phosphonate (Jankowska et al., Tetrahedron Lett. 35, 3355 (1994).
Other methods include oxidative phosphitylation of nucleosides with phosphinic acid in the presence arene sulfonyl derivatives using suitably protected nucleosides. Sekine and Hata, supra. Sekine et al., Tetrahedron Lett. 29, 1037 (1988), used phosphonic acid with mesitylenedisulfonyl chloride, but observed a significant side reaction comprising formation of bisnucleoside H-phosphonate diesters. The result was oxidation of the desired H-phosphonate monoesters by the condensing reagent. Garegg et al., J. Chem. Soc. Perkin Trans. II., pp. 1209-1214 (1987). Replacement of sulfonyl chloride with pivaloyl chloride did not reduce this side reaction.
Stawinski and Thelin, Nucleosides & Nucleotides 9, 129 (1990), found that they could produce the H-phosphonate monoesters almost exclusively if the phosphonic acid is first converted to pyrophosphonate, which can be done in the presence of the nucleoside and condensing reagent.
While there has been much interest and work on the development of methods for fast and efficient oligonucleotide synthesis, improved methods are still desirable.